An overview of tissue and whole organ decellularization processes.

About 2,000 patients now await a donor lung in the United States. Worldwide, 50 million individuals are living with endstage lung disease. Creation of a bioartificial lung requires engineering of viable lung architecture enabling ventilation, perfusion and gas exchange. We decellularized lungs by detergent perfusion and yielded scaffolds with acellular vasculature, airways and alveoli. To regenerate gas exchange tissue, we seeded scaffolds with epithelial and endothelial cells. To establish function, we perfused and ventilated cellseeded constructs in a bioreactor simulating the physiologic environment of developing lung. By day 5, constructs could be perfused with blood and ventilated using physiologic pressures, and they generated gas exchange comparable to that of isolated native lungs. To show in vivo function, we transplanted regenerated lungs into orthotopic position. After transplantation, constructs were perfused by the recipient’s circulation and ventilated by means of the recipient’s airway and respiratory muscles, and they provided gas exchange in vivo for up to 6 h after extubation.

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Regeneration and orthotopic transplantation of a bioartificial lung

The definitive treatment for end-stage organ failure is orthotopic transplantation. However, the demand for transplantation far exceeds the number of available donor organs. A promising tissue-engineering/regenerative-medicine approach for functional organ replacement has emerged in recent years. Decellularization of donor organs such as heart, liver, and lung can provide an acellular, naturally occurring three-dimensional biologic scaffold material that can then be seeded with selected cell populations. Preliminary studies in animal models have provided encouraging results for the proof of concept. However, significant challenges for three-dimensional organ engineering approach remain. This manuscript describes the fundamental concepts of whole-organ engineering, including characterization of the extracellular matrix as a scaffold, methods for decellularization of vascular organs, potential cells to reseed such a scaffold, techniques for the recellularization process and important aspects regarding biore...

https://www.annualreviews.org/doi/pdf/10.1146/annurev-bioeng-071910-124743

Whole-Organ Tissue Engineering: Decellularization and Recellularization of Three-Dimensional Matrix Scaffolds

The definitive treatment for end-stage organ failure is orthotopic transplantation. However, the demand for transplantation far exceeds the number of available donor organs. A promising tissue-engineering/regenerativemedicine approach for functional organ replacement has emerged in recent years. Decellularization of donor organs such as heart, liver, and lung can provide an acellular, naturally occurring three-dimensional biologic scaffold material that can then be seeded with selected cell populations. Preliminary studies in animal models have provided encouraging results for the proof of concept. However, significant challenges for three-dimensional organ engineering approach remain. This manuscript describes the fundamental concepts of whole-organ engineering, including characterization of the extracellular matrix as a scaffold, methods for decellularization of vascular organs, potential cells to reseed such a scaffold, techniques for the recellularization process and important aspects regarding bioreactor design to support this approach. Critical challenges and future directions are also discussed.

Whole-Organ Tissue Engineering: Decellularization and Recellularization of Three-Dimensional

Rationale: Extracellular matrix (ECM) is a dynamic tissue that contributes to organ integrity and function, and its regulation of cell phenotype is a major aspect of cell biology. However, standard in vitro culture approaches are of unclear physiologic relevance because they do not mimic the compositional, architectural, or distensible nature of a living organ. In the lung, fibroblasts exist in ECM-rich interstitial spaces and are key effectors of lung fibrogenesis.Objectives: To better address how ECM influences fibroblast phenotype in a disease-specific manner, we developed a culture system using acellular human normal and fibrotic lungs.Methods: Decellularization was achieved using treatment with detergents, salts, and DNase. The resultant matrices can be sectioned as uniform slices within which cells were cultured.Measurements and Main Results: We report that the decellularization process effectively removes cellular and nuclear material while retaining native dimensionality and stiffness of lung tiss...

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Acellular Normal and Fibrotic Human Lung Matrices as a Culture System for In Vitro Investigation

We report here the first attempt to produce and use whole acellular (AC) lung as a matrix to support development of engineered lung tissue from murine embryonic stem cells (mESCs). We compared the influence of AC lung, Gelfoam, Matrigel, and a collagen I hydrogel matrix on the mESC attachment, differentiation, and subsequent formation of complex tissue. We found that AC lung allowed for better retention of cells with more differentiation of mESCs into epithelial and endothelial lineages. In constructs produced on whole AC lung, we saw indications of organization of differentiating ESC into three-dimensional structures reminiscent of complex tissues. We also saw expression of thyroid transcription factor-1, an immature lung epithelial cell marker; pro-surfactant protein C, a type II pneumocyte marker; PECAM-1/CD31, an endothelial cell marker; cytokeratin 18; alpha-actin, a smooth muscle marker; CD140a or platelet-derived growth factor receptor-alpha; and Clara cell protein 10. There was also evidence of site-specific differentiation in the trachea with the formation of sheets of cytokeratin-positive cells and Clara cell protein 10-expressing Clara cells. Our findings support the utility of AC lung as a matrix for engineering lung tissue and highlight the critical role played by matrix or scaffold-associated cues in guiding ESC differentiation toward lung-specific lineages.

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Andrea Brettler

Papers

Influence of Acellular Natural Lung Matrix on Murine Embryonic Stem Cell Differentiation and Tissue Formation